Isolated human islets require hyperoxia to maintain islet mass, metabolism, and function

https://doi.org/10.1016/j.bbrc.2016.01.110Get rights and content

Highlights

  • Hyperoxia alleviates human islet volume loss during in vitro culture.

  • Hyperoxia maintains human islet viability, metabolism and insulin release function.

  • Optimal partial oxygen pressure for the isolated human islet was clarified.

Abstract

Pancreatic islet transplantation has been recognized as an effective treatment for Type 1 diabetes; however, there is still plenty of room to improve transplantation efficiency. Because islets are metabolically active they require high oxygen to survive; thus hypoxia after transplant is one of the major causes of graft failure. Knowing the optimal oxygen tension for isolated islets would allow a transplant team to provide the best oxygen environment during pre- and post-transplant periods. To address this issue and begin to establish empirically determined guidelines for islet maintenance, we exposed in vitro cultured islets to different partial oxygen pressures (pO2) and assessed changes in islet volume, viability, metabolism, and function. Human islets were cultured for 7 days in different pO2 media corresponding to hypoxia (90 mmHg), normoxia (160 mmHg), and hyerpoxia (270 or 350 mmHg). Compared to normoxia and hypoxia, hyperoxia alleviated the loss of islet volume, maintaining higher islet viability and metabolism as measured by oxygen consumption and glucose-stimulated insulin secretion responses. We predict that maintaining pre- and post-transplanted islets in a hyperoxic environment will alleviate islet volume loss and maintain islet quality thereby improving transplant outcomes.

Introduction

Pancreatic islet transplantation is an effective treatment for Type 1 diabetes and allows patients to be free from insulin injections and uncontrollable hypoglycemic episodes [1], [2], [3]. Currently, isolated islets are infused into the liver through the portal vein; however, more than 50% of injected islets are destroyed because of a hostile microenvironment caused by blood-mediated inflammation reactions, low oxygen tension, or high levels of glucose and toxins [3], [4], [5]. Transplantation of isolated islets at extrahepatic sites face similar issues, and these sites often present poor vascularity and hypoxia [6], [7]. Improved oxygenation to the transplantation site might be a promising solution to prevent islet loss; however, the optimal oxygen tension for isolated islets remains unknown.

In the native pancreas, islets are exposed to a high partial oxygen pressure (pO2: approximately 40 mmHg) and receive more blood flow than the rest of the pancreatic tissue [3], [7], indicating that islets consume more oxygen because of their high metabolic activity [6]. Unlike whole organ transplants which largely preserve capillaries, islets used for transplantations are isolated by enzymatic and mechanical digestion which destroys the islet capillary network [8]. According to current practices, after isolation islets are cultured for a short period under 21% oxygen and 5% CO2 at one atmospheric pressure, and the pO2 of the media is equilibrated to a predicted 160 mmHg. Equilibration and the predicted pO2 value are calculated mathematically without considering the oxygen consumed by active islets. Although the predicted pO2 is four times higher than that reported in the native pancreas, islet central necrosis often develops. This suggests that the oxygen requirement for isolated islets differs from that in the native pancreas possibly due to environmental or structural changes. In this regard, it remains critically important to determine the optimal pO2 that maintains the health of isolated islets pre-transplantation.

Providing the optimal pO2 is also crucial post-transplantation. Regardless of the transplant sites, islets have to rely on the oxygen within surrounding tissue until newly formed microcapillaries can begin to deliver oxygen [3], [7], [9]. However, similar to cultured islets, there is no available information that quantifies the pO2 required for islets to survive post-transplantation. Keeping in mind the need to establish evidence based guidelines, the following in vitro studies were conducted to determine the optimal oxygen concentration for isolated human islets and to identify pO2 levels that promote improved transplantation efficiencies and better patient outcomes.

Section snippets

Isolation of human islets

Human islets were isolated by the Islet Manufacturing Team of the Southern California Islet Cell Resources Center (SC-ICRC) [8], [10]. The use of human tissues in this study was approved by the Institutional Review Board of the Beckman Research Institute.

Human islet culture under different oxygen concentrations

Islets were cultured in a 24-well plate at 250 IEQ/well. Cells were maintained in 1000 μL of RPMI1640 medium containing 5 mmol/L glucose and 10% FBS at 37 °C for 7 days under various oxygen concentrations (10, 21, 35, and 50% oxygen) plus 5% CO2

Simulated and measured values of pO2 in culture medium under various oxygen settings

Simulation results predicted a pO2 gradient within the media as a result of islet oxygen consumption. The simulated pO2 around islets under air containing 21% oxygen were calculated as 152.6, 147.4 and 134.3 mmHg in 300, 500 and 1000 μL of media, respectively (Fig. 1A). In the simulation, OCR also acted to decrease the pO2 (Fig. 1B). The measured average pO2 in 1000 μL of culture media on day 1 under 10, 21, 35 and 50% oxygen was 90, 160, 270 and 350 mmHg, respectively (Fig. 1C).

Cultured islets gradually degrade under 21% oxygen

The physical

Discussion

To date, we have had little direct knowledge of how pO2 influences islet physiology and survival and how these changes affect islet transplantation efficiency. Current practices culture isolated human islets under normoxic conditions (21% oxygen) before transplantation. In contrast, we find that islets cultured under hyperoxic conditions better maintain islet volume, viability, metabolism, and function.

Our simulations imply that culture methods considerably affect pO2 around the islets, which

Acknowledgments

H.K. designed the study, collected data, and wrote the manuscript. D.K., L.M., A.B. and D.M. collected data. J.R., K.O, K.F., Y.T., F.K. and Y.M. reviewed and edited the manuscript.

This work was supported by a grant from the Nora Eccles Treadwell Foundation (30.6990.973667). Human islets were provided by the Integrated Islet Distribution Program (IIDP). We acknowledge the Manufacturing Team led by Dr. Ismail Al-Abdullah at the Southern California Islet Cell Resources Center, Beckman Research

References (19)

There are more references available in the full text version of this article.

Cited by (0)

View full text